2.1. Control of Gene Expression in Transgenic Animals
The production of transgenic animals for both experiment and agricultural purposes is now well known (Wilmut et al., Jul. 7, 1988, New Scientist pp. 56-59). In research, transgenic animals are a powerful tool that have made significant contributions to our understanding of many aspects of biology and have contributed to the development of animal models for human diseases (Jaenisch, 1988, Science 240:1468-1474). It is also clear that several livestock species can be made transgenic and these species promise to expand and revolutionize the method of production and diversity of pharmaceutical products available in the future, in addition to improving the agricultural qualities of the livestock species (Wilmut et al., supra).
A critical, often neglected, aspect of developing transgenic animals is the process whereby expression of the newly introduced gene, referred to as the transgene, is controlled. This is an important process since stringent regulation of transgene expression is often important both for practical, regulatory and safety reasons and to maintain the health of the transgenic animal. In the past either "inducible" or "tissue specific" regulatory mechanisms have been used. Inducible regulation is defined herein as a method of gene regulation which allows for some form of outside manipulation of the onset and/or level of transgene expression. Tissue specific regulation is defined herein as a method for targeting transgene expression to particular tissues or organs.
Inducible gene regulation may be achieved using relatively simple promoter systems such as the metallothionein heat shock promoters, or by using promoters which are responsive to specific compounds such as the Mouse mammary tumor virus LTR which is responsive to glucocorticoid stimulation. More flexible, though more complex inducible regulation systems can be achieved through a "binary" gene approach which utilizes a transactivator gene product to control expression of a second gene of interest. Tissue specific gene regulation usually consists of simple single gene methods (Byrne et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:5473-5477; Ornitz et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88:698-702), although binary transactivator systems can also provide a high degree of tissue specificity.
These current systems provide only a limited ability to control the time of transgene expression within individual animals. In this respect tissue specific promoter elements provide no method to control the onset of transgene activity, but function merely to target gene expression to defined sites. Simple inducible promoters such as metallothionein generally lack tissue specificity and usually have some aspect of endogenous basal expression which cannot be controlled. Thus even for the extensively used inducible metallothionein promoter this approach at best only permits selection of the time at which a relative increase in transgene expression can be induced.
Binary transactivation systems typically consist of two transgenic animals. One animal contains the gene of interest controlled by a promoter element that requires a specific transactivator gene product for expression. Thus, the gene of interest is not expressed in the absence of the transactivator. A second transgenic animal is then made which expresses the required transactivator in the desired tissue. By mating these two transgenic animals, offspring containing both the gene of interest and the transactivator transgene can be produced. Only in these doubly transgenic animals is the gene of interest expressed. Since expression of the gene of interest requires the transactivator, this binary approach dramatically reduces or eliminates any undesirable basal expression inherent in simple inducible systems. Additionally, if expression of the transactivator is targeted using a tissue specific promoter, then in the double transgenics, expression of the gene of interest is in effect targeted to the same specific tissue. Binary systems provide therefore a low resolution method of temporal regulation in as much as they allow the determination of which generation of animals will express the gene of interest. These systems provide little ability, however, to control the time and level of gene expression within an individual transgenic animal.
For many applications it is necessary to accurately control the time and pattern of transgene expression within an individual transgenic animal. For example, many attempts have been made to produce transgenic pigs which express increased levels of growth hormone (Vize et al., 1988, J. Cell Sci. 90:295-300; Pinkert et al., 1990, Dom. Animal Endocrinol. 7:1-18). Elevated growth hormone levels dramatically decrease the amount of body fat in pigs, and increase the animals overall feed efficiency. These effects would be beneficial, both to the consumer who could purchase a leaner, healthier product, and to the producer who can profit from having a more efficient animal. To date however, all attempts to increase the level of growth hormone through production of transgenic pigs have also produced serious pathological conditions which greatly reduce the health of the animals. These pathologies are the direct result of uncontrolled, constitutive expression of growth hormone, since many studies using exogenous hormone administration for short periods of time have not produced pathologies, while still benefiting feed efficiency and fat content. In this situation, a regulatory method to control onset and level of expression from a growth hormone transgene would be extremely useful.